Dynamics of Human Telomerase Holoenzyme Assembly and Subunit Exchange Across the Cell Cycle [RNA]

July 13th, 2015 by Vogan, J. M., Collins, K.

Human telomerase acts on telomeres during the genome synthesis phase of the cell cycle, accompanied by its concentration in Cajal bodies and transient colocalization with telomeres. Whether regulation of human telomerase holoenzyme assembly contributes to the cell cycle restriction of telomerase function is unknown. We investigated the steady-state levels, assembly, and exchange dynamics of human telomerase subunits with quantitative in vivo crosslinking and other methods. We determined the physical association of telomerase subunits in cells blocked or progressing through the cell cycle synchronized by multiple protocols. The total level of human telomerase RNA (hTR) was invariant across the cell cycle. In vivo snapshots of telomerase holoenzyme composition established that hTR remains bound to human telomerase reverse transcriptase (hTERT) throughout all phases of the cell cycle, and subunit competition assays suggest that hTERT-hTR interaction is not readily exchangeable. In contrast, the telomerase holoenzyme Cajal body associated protein, TCAB1, was released from hTR in mitotic cells coincident with TCAB1 delocalization from Cajal bodies. This telomerase holoenzyme disassembly was reversible with cell cycle progression, without change in total TCAB1 protein level. Consistent with differential cell cycle regulation of hTERT-hTR and TCAB1-hTR protein-RNA interactions, overexpression of hTERT or TCAB1 had limited if any influence on hTR assembly of the other subunit. Overall these findings reveal a cell cycle regulation that disables human telomerase association with telomeres while preserving the co-folded hTERT-hTR RNP catalytic core. Studies here, integrated with previous work, lead to a unifying model for telomerase subunit assembly and trafficking in human cells.